Biological system for constructing and testing viral vaccines

ABSTRACT

Disclosed is a novel biological system for 1) the evaluation of potential complications of live virus vaccines prior to actual testing in animals or in the field, and 2) the construction of vaccine vectors in which the ability to grow within specific organs or tissues has selectively been eliminated from the virus.

The subject invention was developed, in part, with funds from NIH grantAl 15722.

CROSS-REFERENCE TO A RELATED APPLICATION

This application is a continuation-in-part of our co-pending applicationSer. No. 262,769, filed Oct. 26, 1988, now abandoned.

BACKGROUND OF THE INVENTION

Post-vaccinial encephalitis and disseminated vaccinia are major concernswith the use of vaccinia virus recombinants as immunization vectors inman. However, because of the efficacy of vaccinia virus recombinants,this virus is still being evaluated in a number of laboratories as apotential live vaccine vector against a wide variety of both human andanimal pathogens. Its potential as a vaccine is evident because of itssuccessful employment in the worldwide eradication of Smallpox. Sincethat time, there have been published a number of studies demonstratingthe suitability of the virus as a vector for the delivery and expressionof foreign antigens. Since these foreign antigens behave as would a geneof the vaccinia vector itself, a significant immune response andsubsequent protection against the antigen, and therefore the infectiousagent of interest, would occur simultaneously with the mounting of animmune response against the vaccinia virus vector itself (Piccini, A.,and E. Paoletti [1986] "The use of vaccinia virus for the constructionof recombinant vaccines," Bioessays, 5:248,252.). Based on pastexperience, there are questions concerning both the safety and theoccasional complications (such as spreading and growth in the brain andnervous system) that can arise from the use of vaccinia as a vaccine.Therefore, if vaccinia virus is to be widely used as a vaccine vectorfor wide scale human or animal use, efforts to design attenuated strainsof virus exhibiting a lesser degree of intrinsic virulence must beundertaken (Brown, F., G. C. Schild, and G. L. Ada [1986] "Recombinantvaccinia viruses as vaccines," Nature 319:549-550).

Over the past 4 or 5 years, there has been a tremendous interest in thepossibility of live vaccine vectors and the specific use of vacciniavirus. Vaccinia virus is a known entity, and has received endorsementfrom the World Health Organization (WHO) for this purpose. Oneoverwhelming reason for this endorsement is the fact that the "coldchain" can be broken. Vaccinia based vaccines do not requirerefrigeration and can be administered in the field by non-skilledpersonnel. The market is world-wide, and the technology described hereis suitable for both human and animal vaccines.

The commercial possibilities are enormous. The use of vaccinia virusbased vaccine technology is appropriate for both human and animalvaccines. Each vaccine construct will have to undergo rigorous testingbefore endorsement and thereafter periodic testing during production. Asimple test system which allows both predictions as to relativevirulence coupled with the possibility of custom designing each andevery vaccine would facilitate the overall process tremendously and filla niche in the field which is currently overlooked. The inventiondescribed herein accomplishes these highly desirable goals.

BRIEF SUMMARY OF THE INVENTION

The subject invention concerns a novel vector system for the assay oftissue tropism and virulence potential of viral vaccines. Specifically,the invention comprises a biological system for (1) the evaluation ofpotential complications of live virus vaccines prior to actual testingin animals or in the field, and (2) the construction of vaccine vectorsin which the ability to grow within specific organs or tissues hasselectively been eliminated from the virus.

The invention vector system is exemplified herein by use ofvaccinia/rabbit poxvirus. This vector system, referred to as the "nullvector system," is shown in FIG. 1 with reference to rabbit poxvirus.This figure shows that the null vector system is comprised of thecomponents:

1. A live rabbit poxvirus deletion mutant, lacking some 30 kilobases ofDNA. This deletion mutant is derived not from vaccinia, but from theclosely related rabbit poxvirus, and it is severely attenuated. Nogrowth of this virus can be detected within any tissue or organ(including the brain) within the animal body except the outer epidermaltissue of the skin. In addition, this mutant exhibits a drasticallyreduced host range in vitro and fails to grow on many cell linesnormally permissive for the wild type virus.

2. A shuttle plasmid, constructed to allow for the efficient, selectiveintroduction of foreign DNA into the deletion mutant.

3. A tissue culture assay system for growth of the virus in vitro thatcomprises marker cell lines that have been correlated with the abilityof the virus to grow within specific tissues and organs within the body.

4. Not shown in FIG. 1 is an in vivo assay system monitoring the weightloss response of test animals, e.g., mice, to recombinant virus. Thepreferred route of administration to the test animal is intracranialinjection.

These components allow for the insertion of DNA into the avirulentdeletion mutant and an interpretation of the effect of the newlyinserted, expressed foreign DNA on in vivo virulence of the construct bythe simple test of growth either on the predictive cell lines, or byadministration to animals. The essential feature of the system, asexemplified herein, involves the novel use of the hemagglutinin genederived from vaccinia virus which has been isolated and cloned into therabbit poxvirus genome. Rabbit poxvirus does not synthesize an activehemagglutinin. The cloning of foreign DNA into the hemagglutinin gene isaccomplished by making use of the shuttle plasmid (FIG. 2) which alsocontains the hemagglutinin gene interrupted by a multicloning site whichallows for simple insertion of the DNA sequences to be tested. Theshuttle plasmid has been designed so that successful insertion of theforeign DNA into rabbit poxvirus tested strain renders the clonedhemagglutinin gene of the virus inactive.

Further, the subject invention also concerns the novel diagnostic use ofknown cells and a sensitive weight loss animal model to asses theeffects and to act as indicators of potential biological complicationwith viral vaccines. Still further, the invention concerns the use ofavirulent poxviruses as a vaccine, and as novel vaccine vectors.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 depicts the rationale for the null vector system.

FIG. 2 shows the application of the null vector system strategy.

FIG. 3 depicts isolation of the vaccinia hemagglutinin gene fromvaccinia strain IHD-J.

FIG. 4 shows construction of hemagglutinin insertion vector.

FIG. 5A shows average weight loss in mice following i.c. injection ofmice with 1×10⁵ pfu of the control tester strain alone (RPμhr23), wildtype virus, and recombinants containing DNA inserted into thehemagglutinin gene of the tester strain by procedures as disclosedherein. Shown are recombinants containing fragments of the wild-typerabbit poxvirus genome missing in the tester strain but replaced in therecombinants that are being evaluated for their contributions tovirulence. Controls include wild type RPV as well as mock infected CEFpurified protein and saline. As can be seen, two of the recombinants(designated μ23HinM and μ23Eco3) cause significant weight loss, which ismeasured at day 4 post inoculation. One of these recombinants, μ23Eco3,restores enough virulence to cause death in the mice, with an LD₅₀ of2×10⁶ pfu. In both cases, the mice show signs of clinical sickness, themagnitude of which corresponds with the relative weight loss. The LD₅₀of RPμhr 23 is>1×10⁸ pfu. The tester strain, RPμ23, as well as severalof the other recombinants (μ23HinN, μ23HinK, and μ23Eco2) do not showclinical signs of sickness or weight loss. This shows the sensitivity ofthis model in detecting even small contributions to virulence by a pieceof insert DNA encoding even a single gene, as in the case of μ23HinM.

FIG. 5B shows dose dependent weight response (percent weight change) ofthe tester strain (RPμhr23) and two of the recombinants whichdemonstrate increased virulence (μ23HinM and μ23Eco3). The relationshipof percent weight loss of each virus strain with change of dosedemonstrates the sensitivity of the weight loss model in detecting evensmall changes in virulence.

DETAILED DESCRIPTION OF THE INVENTION

We have invented a novel vector system which provides a predictive indexconcerning relative virulence and the ability of a given vaccine strainto grow within specific tissues (such as the brain) within the body. Thesystem also opens up the way for the design of vaccine strains of knownand specifically engineered tissue and organ tropism.

The salient feature of the system, as disclosed herein, is that we haveengineered a "new gene" into rabbit poxvirus solely for use as a cloningsite. In wild-type or control strains, this gene, e.g., thehemagglutinin gene, is not expressed. Therefore, by using this gene as acloning site, insertion of foreign DNA inactivates the hemagglutiningene so that any altered effects of growth or pathogenicity can onlyresult from the newly inserted foreign DNA. In addition, the use of thehemagglutinin gene provides a simple colorimetric assay for potentialrecombinants since hemagglutinin positive (HA+) plaques of virus absorbred blood cells whereas hemagglutinin negative (HA-) plaques of virus donot.

Due to the extremely close relatedness of rabbit poxvirus and vaccinia,this system allows for the identification of genetic elementsresponsible for specific tissue tropism and virulence of either virus.Localization of these virulence and tissue tropic associated genesinvolves the systematic reintroduction of sequences deleted in anavirulent tester strain, e.g., RPVμhr23, and then an assay of theresulting recombinants for changes in growth or pathogenicity in thetest system. Once these genes have been localized, they can beselectively eliminated from any vaccine strain, if desired. Likewise,this system can be used to detect any unanticipated alterations ingrowth of any newly-designed vaccine construct, thus greatly aiding inthe development of new recombinant vaccines. This system also can beroutinely used for "batch analysis⃡ of production vaccines, thusproviding a quick and cost-efficient method to insure that vaccine lotsare uniform, and predictably avirulent, based simply on the ability togrow on well-defined indicator cell lines.

Any avirulent member of the poxvirus family can be used so long as it iscapable of sufficient expression of an immunogen to sensitize the host.Thus, the avirulent poxvirus can be a deletion mutant capable of growth.

The poxvirus can be modified, in accord with the invention, by insertinga suitable marker gene, e.g., the hemagglutinin gene derived fromvaccinia virus. Though the hemagglutinin gene is disclosed herein toexemplify the invention, any prokaryotic or eukaryotic gene whoseexpression and subsequent inactivation which could be selected for canbe used. For example, the bacterial gene encoding luciferase orβ-galactosidase can be used. The modification of the poxvirus can beconducted using standard procedures well known in the art, and as shownin FIG. 2 of the drawings. General techniques to produce modified virusare disclosed in U.S. Pat. No. 4,603,112 (Paoletti et al.).

As shown in FIG. 2, the shuttle vector (plasmid) containing the gene ofinterest (and the marker or indicator gene), flanked by suitablepoxvirus sequences, will undergo recombination with poxvirus whichresults in the integration of the flanked genes into the poxvirusgenome. This recombination occurs in a eukaryotic host cell. The cellsare initially infected with a poxvirus and then transfected with the DNAvector. Infection of eukaryotic cells is by standard well-knownprocedures. Following infection and transfection, the cells areincubated under standard conditions to allow for virus replication atwhich time in vivo recombination occurs between the homologous poxvirussequences in the shuttle vector and the poxvirus sequences in thegenome.

Selection of recombinant viral progeny can be done by standardprocedures well known in the art, as disclosed previously. For example,if the hemagglutinin gene is used as the marker gene, a simplecolorimetric assay can be employed to identify the presence ofsuccessful recombinants. Another marker or indicator gene which can beused in the E. coli lacZ gene encoding β-galactosidase. Recombinantviruses expressing β-galactosidase can be selected for by using achromogenic substrate for the enzyme.

Vaccines comprising live recombinant viruses expressing immunogenicproteins, e.g., rabies virus glycoprotein, herpes simplex virus type 1glycoprotein B, and Epstein-Barr virus glycoprotein gp340, can be usedto vaccinate humans and animals (See Moss, B. and C. Flexner [1987]"Vaccinia virus expression vector," Ann. Rev. Immunol. 5:305-324). Suchvaccines can be administered intradermally, as was done for smallpoxvaccination. Other routes of administration can be used, if desired,depending on the protection sought for any particular disease.

Following are examples which illustrate procedures, including the bestmode, for practicing the invention. These examples should not beconstrued as limiting. All percentages are by weight and all solventmixture proportions are by volume unless otherwise noted.

EXAMPLE 1 Construction of RPμhr23 HA⁺ Recipient Strain

RPμhr23 is a well-known, spontaneously occurring white pock mutant ofwild-type rabbitpox virus (Utrecht strain) that resulted from thepassage of wild-type virus on the chorioallantoic membrane of a chickenegg. This isolate was first described by Fenner and Sambrook (Fenner, F.and J. F. Sambrook [1966] Virology 28:600-609).

Since RPμhr23 does not have a functional hemagglutinin gene, RPμhr23 HA⁺was constructed by introducing a functional hemagglutinin gene into theRPμhr23 (HA⁻) parental strain. This construction involved two steps: 1)isolating a functional HA gene from the known vaccinia virus strainIHD-J (HA⁺) and 2) introducing it into the RPμhr23 (HA⁻) by homologousrecombination to form the RPμhr23 (HA⁺) recepient strain. The stepsinvolved in the construction are outlined below.

1. Isolation of the vaccinia IHD-J hemagglutinin gene

Vaccinia IHD-J was obtained from the American Type Culture Collection(ATCC VR-156). This strain of vaccinia was chosen as the source for thehemagglutinin gene because this gene had been previously mapped andsequenced (Shida, H. [1986] Virology 150:451-462), thus facilitatinggenetic manipulation and its use as an insertional vector. Thehemagglutinin gene was subcloned from vaccinia (see FIG. 3) by digestingthe purified vaccinia DNA with the restriction endonucleases HindIII andEcoRI (New England Biolabs). The resulting fragments of this doubledigest were resolved by electrophoresis on a 0.5% agarose gel inTris-Borate buffer (TBE; 89 mM Tris base, 89 mM Boric acid, 30M Na₂EDTA; pH 8.3) at 50 V for 2 hr. The 7.8 kb fragment containing thehemagglutinin gene was easily resolved, and excised. The DNA waselectroeluted using the IBI electroelutor. This fragment can besubcloned into pUC 9 (Pharmacia, Inc.) and transformed into bacterialstrain E. coli UT481. Other strains such as E. coli DH5α(BRL) can beused. The resulting plasmid which contained the EcoRI/HindIII fragmentcontaining the functional hemagglutinin gene was designated pHGN. Inorder to minimize the amount of extraneous viral sequences flanking thehemagglutinin gene, pHGN was digested with the restriction endonucleasesHindIII and SaII which excise a 1800 bp fragment with contains thevaccinia hemagglutinin gene and approximately 400 bp of viral flankingsequence on either side of the coding region for the hemagglutinin geneproduct. This 1800 bp fragment was excised from the gel andelectroeluted as described previously. The resulting DNA was thentreated with T4 DNA polymerase (International Biotechnologies) in orderto produce flush ends. Synthetic KpnI linkers were then added, andligated to the fragment. The resulting mixture was then subjected todigestion with KpnI restriction endonuclease, and the cut linkerspurified away through a Sephadex G-50 spun column. The resulting 1806 bpfragment, which consisted of the isolated hemagglutinin fragment and theKpnI linkers on each end, was ligated to pUC 9 which had been digestedwith KpnI. The ligated mixture was then used to transform E. coli UT481,and the resulting recombinant was designated pHGN1. The importantfeatures of this plasmid is that if contained the 988 bp hemagglutiningene, and a total of 853 bp of flanking sequence, approximately 400 oneach end. The gene was cloned into pUC 9 using KpnI linkers to aid inexcising the gene for future manipulation. The flanking sequence allowedthe hemagglutinin to recombine into rabbit poxvirus.

2. Transfection of PRμhr23 (HA-) with pHGN1 to yield RPμhr23 (HA+)recipient strain.

The parental strain of RPμhr23 (HA⁻) was transfected with the pHGN1plasmid by a modification of a commonly used procedure (Condit, R. C.,A. Motyczka, and G. Spizz [1983] Virology 128:429-443). Primary chickenembryo fibroblasts (CEF) were grown in medium 199 (Gibco) supplementedwith 10% fetal bovine serum, 3 mg/ml tryptose phosphate broth, 2 mMglutamine, 100 units penicillin, 100 mg streptomycin, and 0.1 mgpyruvate per ml. For the transfection, media was aspirated from aconfluent monolayer of primary chicken embryo fibroblasts, and waswashed twice with phosphate-buffered saline (PBS; 0.01M sodium phosphateplus 0.15M NaCl [pH7.2]), and infected with RPμhr23 (HA⁻) at amultiplicity of infection (m.o.i.) of 0.05 in unsupplemented media. Thevirus was allowed to adsorb for two hr by incubating at 37° C., 5% CO₂with humidity. During this incubation, 40 μg of pHGN1 plasmid DNA wasdiluted with 1×HEPES Buffered Saline (20 mM HEPES, 150 mM NaCl, 700M Na₂HPO₄, 5 mM KCl, 6 mM Glucose) to a final volume of 4 ml. A pasteur pipetwas then used to cavitate the DNA solution while 1/20 volume (200 μl) of2.5M CaCl₂ was added. This mixture was then allowed to precipitate atroom temperature for 1 hour. At the appropriate time, the infectinginoculum was removed from the CEF monolayer, and the cells washed twotimes with unsupplemented medium. 4 ml of the precipitated DNA was thenadded to the monolayer and incubated for 30 min at room temperature.After this incubation, 40 ml of supplemented media was added to themonolayer and incubated 3.5 hr at 37° C. After this time period, themedia was aspirated, and fresh media added. The monolayer was thenincubated for 48 hr at 37° C. The infected cells were then harvested byscraping with a rubber policeman, and concentrated by centrifugation at8000×g for 20 min. The infected cell pellet was resuspended in 10 ml PBSand briefly sonicated with a probe sonicator to disrupt the cells andrelease the virus. In order to identify the HA⁺ recombinant, thetransfection mixture was plaqued on QT-6 quail cells at medium density,overlayed with complete medium containing 1% methylcellulose, and grownfor 2 days. The HA⁺ recombinants were visualized using a hemabsorptionassay. Briefly, the methylcellulose was aspirated from the dishes, andthe monolayers washed 3 times with PBS to remove the residualmethylcellulose. 15 ml of a 0.5% suspension of washed chicken red bloodcells were added to the monolayer, and the monolayer incubated at 37° C.for 15 min. The red blood cell suspension was then removed, and the HA⁺plaques could be seen by their red color. Several of these plaques werepicked and purified by three rounds of plaque purification.

EXAMPLE 2 Construction of the Insertion Plasmid pHGN3

In order to facilitate the insertion of cloned DNA into thehemagglutinin gene of RPμhr23 (HA⁺), plasmid pHGN1 was modified by theremoval of extraneous pUC sequences, and the addition of a multicloningsite which generates 6 unique restriction sites within the HA gene. Thisallows greater flexibility in cloning DNA into this gene. Theconstruction of this plasmid is shown in FIG. 4 and outlined below.

1. Removal of the lac Z from pUC

The first step of the construction involved the removal of the lacZ geneand internal polylinker from pUC 9. pUC 9 was digested with therestriction endonuclease HaeII, which liberates two fragments, one ofwhich contains the lacZ cassette and polylinker, and the other whichcontains sequence essential for the replication of the plasmid. Thisrestriction digest was ligated, transformed into E. coli, andβ-galactosidase negative bacteria were selected by the addition of X-galand IPTG to the selection medium. This plasmid was isolated and shown tolack the lacZ cassette fragment. The 1800 bp hemagglutinin gene wasisolated from pHGN1, and ligated into this modified pUC plasmid by theaddition of KpnI linkers to the plasmid. The resulting plasmid wasdesignated pHGN2.

2. Insertion of the multicloning site

The multicloning site was obtained by digesting pUC 9 with therestriction endonucleases HindIII and HaeIII, and isolating theresulting 47 bp multicloning site by electrophoresis on a 4% Nusieve gel(FMC Bioproducts). The fragment was electroeluted from the agar andtreated with T4 DNA polymerase to produce blunt ends. The fragment wasthen inserted into pHGN2 that had been digested with the endonucleaseAccI, which cleaves at base 43 of the 5' coding region of thehemagglutinin gene. The resulting plasmid was designated pHGN3, andallows one to easily clone DNA fragments into the hemagglutinin gene,which then acts as a shuttle to introduce them into the RPμhr23 vector.

EXAMPLE 3 Assay of RPμhr23 Recombinants for Changes in VirulenceProperties

Once the RPμhr23 recombinants containing the DNA fragments of interesthave been constructed and isolated, changes in virulence resulting fromthe inserted DNA fragment can be ascertained. A two-component assaysystem has been developed to detect changes in virulence and tissuetropism. The first component involves the testing of the recombinantvirus for changes in growth characteristics on a number of marker tissueculture cell lines in vitro. The cell lines selected have been shown tobe predictive of certain growth characteristics within the animal. Thesecond assay involves in vivo testing of the recombinants in the mousefor changes in LD₅₀ (death) or for causing sickness (i.e., weight loss).Here is described the application of these tests. The number of celllines employed can be modified, and the use of substitute cell lineswith similar properties is possible. Also, while we describe only oneroute of inoculation (intracranial--i.c.) of the virus into the mice,other routes (e.g., intraperitoneal--i.p.) may be used as well. Theassay criteria for sickness is herein described; however, otherindicators, such as elevated body temperature, can also be used. Thesetwo virulence assays can be used individually or in combination.

1. Assay for changes in host range in marker cell lines in vitro

Sufficient quantities of virus for testing are grown up on chickenembryo fibroblasts (CEFs) by standard procedures (and as describedabove). Changes in growth on the marker cell lines are determined byplaquing various dilutions of the virus on a panel of cell lines whichwe have shown to discriminate between various levels of virulence in theanimal. These cell lines represent continuous lines of cells ofdifferent tissue and species origin. In a representative form of theassay, dilutions of the virus ranging from 1 to 10,000 pfu (asdetermined on the permissive avian cell lines) are added to confluentmonolayers of the test lines in 60 mm dishes. The virus is allowed toadsorb for two hours, and then the monolayers are overlayed with 5 ml ofsemi-solid medium, typically 1×medium that contains either 0.75% agaroseor 1% methylcellulose. The monolayers are then incubated for three days,at which time they are assayed by the addition of a vital dye, such asneutral red. The recombinants are evaluated either for the ability toform plaques as evidence of productive infection, or by changes in thecytopathic effects of the virus at the higher multiplicities ofinfection. The assay is performed with appropriate control viruses, suchas RPμhr23 (which can grow only on the avian marker cell lines and whichis avirulent in the mouse) and several other mutants with deletions ofdiffering size which retain the ability to grow on one or moreadditional cell lines and which, in turn, show different virulencecharacteristics in the mouse. It is then possible to predict changes inthe virulence properties of a given newly generated recombinant. Thisassay can be modified using substitute cell lines. Rather than a plaqueassay, an end-point dilution type assay in a microtiter dish could be anappropriate substitute. An endpoint dilution assay could be monitoredsimply by changes in growth characteristics of the monolayer or by aELISA reader.

2. Assay for changes in vivo using a sensitive weight loss model in themouse

Since the properties governing virulence are complex, it is possiblethat a recombinant may not cause an increase in host range in cellculture, but be capable of causing sickness or death when administeredto animals. The following assay is a sensitive complementary method tothe in vitro assay discussed above for evaluating a recombinant for theability to cause death or sickness. Here we describe an assay based on aweight loss model as an objective and sensitive assessment of sicknessresulting from changes in virulence. The particular example citedresults from insertion of a single fragment of DNA into the testerstrain RPμhr23.

Sufficient quantities of the virus are grown up for testing as describedabove. Virus was purified for injection into mice essentially asfollows: Typically infected cells from 10-150 mm dishes that had beeninfected at m.o.i. of 0.05-0.1 and harvested after 4 days at 37° C. werecollected and resuspended in 10 ml of PBS. The virus was released fromthe cell debris by two rounds of freeze-thawing, followed by sonicationfor 30 seconds to one minute in a water bath sonicator. This mixture wasthen overlayed on a 5 ml 50% sucrose cushion in a Beckman SW27 tube, andbrought up to volume (approx. 38 ml) in PBS. The virus was pelleted at58,000 xg at 4° C. for 90 minutes. The supernatant was aspirated and thepellet drained and resuspended in 1.5 ml of PBS. The virus wasresuspended using a chilled water bath sonicator, and layered onto a 36ml linear 20-50% potassium tartrate gradient. Following centrifugation,for 60 minutes at 58,000 xg, the viral band was collected and diluted inPBS (at least 3 volumes). The virus was then pelleted throughcentrifugation at 90 minutes at 79,000 xg at 4° C., and resuspended in500 μl-1 ml of PBS by sonication and repeated pipetting. This stock wastitered and used neat or diluted in PBS for injection into mice.

Four to six week-old female BaLB/c mice (Sasco's pathogen-free colony)were maintained five to six mice per cage in an isolation unit. Forintracerebral (i.c.) inoculations, virus (undiluted and diluted in PBS)was injected into the left cerebral hemisphere using a 27 gaugetuberculin syringe. Typical ranges of inoculum are from 20 pfu to 1×10⁷pfu. The mice were weighed just prior to injection and on each daythereafter. The mice were also watched closely for signs of illness anddeath. Weight loss was monitored for at least 6 days or until mice showeither a positive weight gain for two or more subsequent days or untildeath. The LD₅₀ was determined by standard procedures. FIGS. 5A and 5Billustrate the sensitivity of this assay.

As can be seen, while RPμhr23 causes no change in virulence in theanimal, several of the recombinants cause a significant amount of weightloss (up to 48% of the original body weight). Significantly, we haveshown that this weight loss correlates with lethality, and that theweight loss is a much more sensitive assay for virulence than is LD₅₀alone. Also significant is that the weight loss in several of therecombinants occurs prior to any predicted increase in virulence asmeasured by our in vitro cell culture assay. Therefore, we feel that theweight loss model is a sensitive assay that complements, but does notnecessarily replace, the cell culture system as a means by which toevaluate contributions to virulence of a single gene or groups of genes.

Biological materials which can be used according to the subjectinvention have been deposited in the permanent collection of theAmerican Type Culture Collection (ATCC), 12301 Parklawn Drive,Rockville, Md. 20852 USA. The accession numbers and deposit dates are asfollows:

    ______________________________________                                        Culture          Repository No.                                                                            Deposit Date                                     ______________________________________                                        Escherichia coli pHGN3                                                                         69051       August 17, 1992                                  Rabbitpox virus RPμhr23HA.sup.+                                                             VR 2379     August 13, 1992                                  ______________________________________                                    

The subject cultures have been deposited under conditions that assurethat access to the cultures will be available during the pendency ofthis patent application to one determined by the Commissioner of Patentsand Trademarks to be entitled thereto under 37 CFR 1.14 and 35 USC 122.The deposits are available as required by foreign patent laws incountries wherein counterparts of the subject application, or itsprogeny, are filed. However, it should be understood that theavailability of a deposit does not constitute a license to practice thesubject invention in derogation of patent rights granted by governmentalaction.

Further, the subject culture deposits will be stored and made availableto the public in accord with the provisions of the Budapest Treaty forthe Deposit of Microorganisms, i.e., they will be stored with all thecare necessary to keep them viable and uncontaminated for a period of atleast five years after the most recent request for the furnishing of asample of the deposit, and in any case, for a period of at least 30(thirty) years after the date of deposit or for the enforceable life ofany patent which may issue disclosing the cultures. The depositoracknowledges the duty to replace the deposits should the depository beunable to furnish a sample when requested, due to the condition of thedeposit(s). All restrictions on the availability to the public of thesubject culture deposits will be irrevocably removed upon the grantingof a patent disclosing them.

We claim:
 1. A biologically pure culture of a rabbit poxvirus denotedRPμhr23 HA⁺.
 2. Plasmid pHGN3.